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[Cancer Research 60, 1084-1091, February 15, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

Transcriptional Regulation of Cyclooxygenase-2 Gene Expression: Novel Effects of Nonsteroidal Anti-Inflammatory Drugs

Chiun-Jye Yuan1, Asim K. Mandal1, Zhongjian Zhang and Anil B. Mukherjee2

Section on Developmental Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892-1830


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenase-2 (COX-2) gene overexpression is suggested to play important roles in colorectal tumorigenesis. Epidemiological studies revealed that nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and sulindac, which inhibit COX activity, reduce colorectal cancer mortality. Current investigations have focused on delineating the molecular mechanisms that regulate COX-2 gene expression and the roles of NSAIDs in cancer chemoprevention. COX-2 catalyzes the production of prostaglandins (PGs) from arachidonic acid (AA), generated by phospholipases A2 (PLA2s), a family of acyl esterases that cause the release of AA from cellular phospholipids. Pancreatic secretory PLA2 (sPLA2), via its receptor (sPLA2R), transcriptionally activates COX-2 gene expression in several cell types, although a specific transcription factor mediating COX-2 expression has not yet been identified. Here, we report that a transcription factor, CCAAT/enhancer-binding protein ß (C/EBPß), plays a critical role in sPLA2IB-induced, receptor-mediated COX-2 gene expression in MC3T3E1 and NIH3T3 cells. Furthermore, treatment of these cells with NSAIDs in the presence of sPLA2IB appears to potentiate the stimulatory effects on COX-2 mRNA and COX-2 protein expression and a concomitant elevation in PG production. Most significantly, NSAID treatment appears to drastically suppress the production of cytosolic PLA2 (cPLA2) mRNA. The lack of sPLA2IB, sPLA2IIA, and sPLA2V mRNA expression in both NIH3T3 and MC3T3E1 cells suggests that cPLA2 is the most likely enzyme that catalyzes the release of AA, the rate-limiting substrate of COX for the production of PGs. Our results suggest that: (a) sPLA2IB receptor-mediated COX-2 expression is mediated via C/EBPß; (b) NSAIDs in the presence of sPLA2IB potentiate the stimulatory effects of sPLA2IB on COX-2 mRNA expression; and (c) despite the apparent stimulation of COX-2 expression by NSAIDs, they strikingly deprive COX-2 of its substrate, AA, by suppressing cPLA2 mRNA expression. Both AA and PGs regulate many vital biological functions (e.g., motility and invasiveness) that are dysregulated in most cancer cells, and they have profound effects on cellular differentiation. Our results raise the possibility that deprivation of COX-2 of its substrate by the suppression of cPLA2 mRNA expression is an additional mechanism used by NSAIDs to inhibit tumorigenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Investigations on the mechanism of action of aspirin uncovered COX3 as the key enzyme responsible for prostanoid production (reviewed in Ref. 1 ). Also known as PG endoperoxide synthase (EC 1.14.99.1), COX is the rate-limiting enzyme for PG production (2 , 3) . Among its two isoforms, COX-1 is expressed constitutively (4) , whereas COX-2 is inducible (5) . Overexpression of the COX-2 gene is suggested to play an important role in promoting colorectal cancer (reviewed in Ref. 6 ). Moreover, the results of several epidemiological studies have shown that NSAIDs, such as aspirin and sulindac, which inhibit COX activity, significantly reduce colorectal cancer mortality (7, 8, 9, 10, 11, 12, 13, 14) . However, neither the mechanism(s) of transcriptional regulation of the COX-2 gene expression in cancer cells nor the cancer-chemopreventative effects of NSAIDs are as yet clearly understood (reviewed in Ref. 6 ). Thus, attempts to identify specific transcriptional mediators that regulate COX-2 gene expression and the mechanism(s) by which NSAIDs modulate tumorigenesis are the focus of current investigations in many laboratories.

PLA2s (EC 3.1.1.4) are a family of enzymes that catalyze the hydrolysis of fatty acyl ester bond at the Sn-2 position of the glycerophosphocholine molecule, generating free fatty acid, such as AA, and lysophospholipid (15 , 16) . AA is further metabolized by COX to produce PGs and thromboxanes (3) . The mammalian sPLA2s have been classified into five different groups: sPLA2IB, IIA, IIC, V, and X (17) . The properties shared by these sPLA2s include their relatively low molecular mass, the presence of several internal disulfide bridges (15) , and a preference for phospholipids with different polar head groups and fatty acid chains. The isolation and characterization of the sPLA2R (18 , 19) have changed the previously held notion that pancreatic PLA2 (sPLA2IB) is solely a digestive enzyme. In fact, it has been demonstrated that, via this receptor pathway, sPLA2IB transcriptionally regulates COX-2 gene expression in MC3T3E1 (20) and rat mesangial (21) cells. Recently, it has been reported that both the sPLA2IB and IIA are natural ligands of the mouse M-type sPLA2R (22) . However, a specific transcription factor that regulates the sPLA2IB-induced COX-2 gene expression, to our knowledge, has not been identified.

In the present study, we sought to: (a) identify the specific factor(s) that regulate the sPLA2R-mediated transcriptional activation of COX-2 gene expression; and (b) determine whether NSAIDs, in addition to their inhibitory action on COX activity, have other effects that alter PG production. Our results show that the nuclear factor C/EBPß plays a critical role in the regulation of sPLA2R-mediated transcriptional activation of the COX-2 gene and further demonstrate that although NSAIDs stimulate the expression of COX-2 mRNA in NIH3T3 cells, they drastically suppress cPLA2 mRNA production. To our knowledge, this is the first demonstration that NSAIDs inhibit cPLA2 mRNA expression and, as a result, deprive COX-2 of its substrate, AA. Because we found that a low level expression of cPLA2 m RNA but not sPLA2IB, sPLA2IIA, and sPLA2V mRNAs is detectable in these cells, cPLA2 is the most likely enzyme that catalyzes the release of AA that is used by COX-2 for PG production. Thus, our observation that NSAIDs drastically suppress cPLA2 mRNA production suggests that these drugs critically restrict the supply of COX-2 substrate, AA, and consequently, inhibit PG production. Furthermore, it is well established that both AA and PGs regulate important biological functions, such as motility, ECM invasiveness, and differentiation, all of which are dysregulated in cancer cells. We propose that the previously reported antimetastatic and cancer-chemopreventative effects of NSAIDs, at least in part, are attributable to their ability to reduce AA production by suppressing cPLA2 mRNA expression, in addition to their well-known inhibitory effects on COX-2 activity, drastically reducing the level of PGs.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
[{gamma}-32P]ATP, [{alpha}-32P]dCTP, Hybond-N+ nylon membrane, Sequenase version 2.0 DNA sequencing kit, and Moloney murine leukemia virus reverse transcriptase were obtained from Amersham (Piscataway, NJ). Fetal bovine serum was from Hyclone and {alpha}MEM, penicillin G, streptomycin, and glutamine were from Biofluids (Gaithersburg, MD). The MC3T3E1 cell line was kindly provided by Dr. Barid Mukherjee (McGill University, Montreal, Quebec, Canada). NIH3T3 cells were from American Type Culture Collection. NS-398 was purchased from Calbiochem (San Diego, CA). Anti-C/EBP-{alpha}, -ß, and -{gamma} IgGs were kindly provided by Dr. Janice Chou (National Institute of Child Health and Human Development, National Institutes of Health, NICHD/NIH, Bethesda, MD). Antibodies for AP2, NF-{kappa}B, p50, and p65 were from Santa Cruz Biotechnology (Santa Cruz, CA). Porcine pancreatic PLA2 (sPLA2IB) was purchased from Boehringer Mannheim (Indianapolis, IN). RNAzol B was from Tel-Test, Inc. (Friendswood, TX), and poly(deoxyinosinic-deoxycytidylic acid) was from Pharmacia (Piscataway, NJ). Amplitaq DNA polymerase, PCR buffer, and other reagents for PCR from Perkin-Elmer (Branchburg, NJ); random priming (Prime-It RmT kit) and QuikChange Site-Directed Mutagenesis kits were from Stratagene (La Jolla, CA); Ste Select-D G-50 spin column was from 5 Prime-3 Prime (Boulder, CO). Oligonucleotide primers for reverse transcription-PCR were custom synthesized by Life Technologies, Inc. (Gaithersburg, MD). COX-2 antibody was purchased from Cayman Chemical (Ann Arbor, MI).

Cell Culture and RNA Isolation.
NIH3T3 and MC3T3E1 cells were maintained in DMEM supplemented with 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 4 mM glutamine at 37°C in a humidified incubator with an atmosphere of 5% CO2 and 95% air. After the cells reached 70–80% confluence in 75-cm2 tissue culture flasks, they were washed with 1x PBS, and then the medium was changed to Opti-MEM 1 containing 5% FBS, 4 mM glutamine, 1 mM CaCl2, and the antibiotics mentioned above. In some cultures, 50 nM porcine sPLA2I or 150 µM aspirin/sulindac (dissolved in DMSO) was added, and the cells were incubated for 6 h. Total RNAs were isolated from the treated and control cells using RNAzol B reagent, following the supplier’s protocol.

Northern Blot Analysis.
Thirty µg of the total RNA were resolved by electrophoresis on a formaldehyde-containing 1.2% agarose gel, transferred onto Hybond-N+ nylon membrane, and cross-linked with UV to the membrane using a Stratalinker (Stratagene). cDNAs of murine sPLA2IB, sPLA2IIA, sPLA2V, cPLA2, COX-2, GAPDH, and ß-actin were labeled with [{alpha}-32P]dCTP using a random priming kit. Unincorporated nucleotides were removed by using a Sephadex G-50 spun column. After denaturation, the labeled probes were hybridized with the RNA blots for 2 h at 68°C using ExpressHyb solution (Clontech, Palo Alto, CA). Specific bands hybridizing with the probes were detected by autoradiography. The amount and the quality of the RNA loaded were monitored by hybridization with the GAPDH or the ß-actin probes after removal of the previously hybridized probes by boiling the blots in 0.5% SDS for 10 min.

Determination of PGE2 Levels.
MC3T3E1 cells were plated in 12-well plastic dishes (9 x 104 cells/well) with 1 ml of {alpha}MEM containing 10% fetal bovine serum and grown to confluence. Cells were then washed three times with PBS, and the media were changed to 0.5 ml {alpha}MEM containing 0.1% BSA supplemented with 50 nM porcine PLA2-I with or without NS-398, a specific inhibitor of COX-2 activity. At indicated time points, the culture medium was withdrawn and diluted 20-fold with water. The amount of PGE2 in media was determined by an ELISA kit. The protein concentration of cell lysate from each well was determined by using the Bio-Rad protein-assay reagent and used to normalize the PGE2 concentration. The results are the mean of two determinations each.

Western Blotting.
Cells were washed once with PBS and collected by scrapping with a rubber policeman. Cell lysates were prepared by incubating the cell pellets with lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP40, and 0.2% deoxycholate] containing 1 mM PMSF and 10 mg/ml each of aprotinin, leupeptin, and pepstatin on ice for 15 min. All solutions were centrifuged, and undissolved residues were discarded. Equal amounts (50 µg) of cell lysate proteins were resolved by electrophoresis on a precast 4–20% gradient SDS-polyacrylamide gel. Protein bands were transferred to a PROTRAN nitrocellulose membrane (Schlercher & Schuell, Keene, NH) and blocked with 5% BSA in PBS at room temperature for 2 h. The membrane was then incubated with polyclonal antibody against COX-2 (1:1000), followed by a horseradish peroxidase-conjugated secondary antibody against rabbit IgG (1:5000). The COX-2 protein band was then visualized by ECL reagent, followed by exposure to Kodak X-ray film.

Construction of Luciferase Reporter Plasmids.
The DNA fragments covering different lengths of the promoter region of the mouse COX-2 gene was subcloned by PCR from genomic DNA extracted from mouse embryonic stem cells, and the nucleotide sequences were confirmed by DNA sequencing. The subcloned promoter regions were then inserted into the promoterless luciferase reporter vector, pGL-Basic (Promega Corp., Madison, WI), between the NheI and HindIII sites. The mutations of AP2 and C/EBPß sites in the COX-2 promoter region were performed on luciferase reporter plasmid containing COX-2 promoter region -188 to 70 bp by using the QuickChange site-directed mutagenesis kit according to the manufacturer’s instructions.

Site-directed Mutagenesis.
Site-directed mutagenesis was used to generate point mutations in the COX-2 promoter region. Two site-directed mutations, mu-AP2 in which the AP2 element (CCGCTGCGG) was mutated to (CCGCTttGG), and mu-C/EBPß, in which the C/EBPß element (TTGCGCAAC) was mutated to (TgGaaCAAC), were constructed using the QuickChange site-directed mutagenesis kit according to the manufacturer’s instructions. Briefly, the luciferase reporter plasmid containing the COX-2 promoter region was denatured and annealed with the primers containing the mutation for AP2 or C/EBPß, respectively. After PCR amplification, the product was treated with endonuclease DpnI and then used to transform XL1-Blue supercompetent cells. The mutated plasmids were screened and confirmed by DNA sequencing analysis.

Transfection of Plasmids and Luciferase Assay.
MC3T3E1 cells (1.1 x 105 cells/well) were cultured on six-well plastic plates to ~65% confluence before transfection. In each well, cells were treated with 1.5 µg of luciferase expression vector containing COX-2 promoter and 9 ml of LipofectAMINE in 1 ml of serum-free medium, Opti-MEM 1 (Life Technologies) at 37°C for 6 h, according to the manufacturer’s instruction. The medium was replaced with 2 ml of {alpha}MEM containing 10% fetal serum albumin. The transfected cells were further grown for 2 days. Medium was replaced with {alpha}MEM containing 0.1% BSA for 18–24 h before incubating with 50 nM porcine sPLA2IB in the same medium at 37°C for 6 h. After incubation, cells were lysed, and luciferase activity was determined in cell lysates by use of the Lumate LB9507 Luminometer using the luciferase assay kit from Promega. The luciferase activity was normalized, based on the protein concentration of each cell lysate.

Preparation of Nuclear Fraction.
MC3T3E1 cells (6 x 105 cells/dish) were plated in 100-mm plastic dishes with 10 ml of {alpha}MEM containing 10% fetal serum albumin and grown to confluence. The medium was replaced with {alpha}MEM containing 0.1% BSA at 37°C for 18–24 h, followed by adding 50 nM sPLA2IB at 37°C for 20 min. After incubation, cells were collected by scrapping, and the nuclear extracts were prepared as described previously (23) . Briefly, the cell pellet was resuspended in 400 µl of cell lysis buffer [10 mM Tris-HCl (pH 7.9), 10 mM KCl, and 0.1 mM each EDTA and EGTA] containing 1 mM PMSF and DTT on ice for 15 min. Then 25 µl of 10% NP40 were added to the cell suspension and vigorously vortexed for 10 s. Nuclei were collected by brief centrifugation. The nuclear fraction was extracted by 50 µl of extraction buffer [20 mM Tris-HCl (pH 7.9), 0.4 M NaCl, and 1 mM each of EDTA, EGTA, DTT, and PMSF] and mixed by constant shaking at 4°C for 15 min, spun at 4°C to remove unextractable residues.

Electrophoretic Mobility Shift Assay.
Three double-stranded DNA probes were prepared by annealing the sense and antisense oligonucleotides of corresponding nucleotide sequences covering three areas of the COX-2 promoter. The double-stranded DNA probes were then end-labeled with [{gamma}-32P]ATP by using T4 polynucleotide kinase. The binding of nuclear proteins (2.5 µg) to the probes (15,000 cpm) was performed in 10 µl of binding buffer [10% glycerol, 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2.5 mM MgCl2, 1.25 mM EDTA, and 1.25 mM DTT] containing 0.5 µg of poly(deoxyinosinic-deoxycytidylic acid) at 30°C for 40 min. For the supershift assay, each antibody (i.e., C/EBP{alpha}, C/EBPß, C/EBP{delta}, NF-{kappa}B, p50, NF-{kappa}B p65, and AP-2) was added into the protein and DNA mixture and incubated overnight at 4°C. After incubation, the mixtures were resolved by electrophoresis on a 4% nondenaturing polyacrylamide gel and autoradiographed.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of COX-2 by Pancreatic sPLA2IB.
To understand the molecular mechanism(s) of sPLA2-R-mediated transcriptional activation of the COX-2 gene, we first studied MC3T3E1 cells and established that sPLA2IB treatment of these cells for a period of 6 h stimulates the expression of COX-2 mRNA and COX-2 protein by Northern (Fig. 1A)Citation and Western (Fig. 1B)Citation blot analyses, respectively. The presence of the sPLA2R in these cells were determined by performing 125I-labeled sPLA2-I binding and affinity cross-linking studies (data not shown). Upon stimulation with sPLA2-IB, these cells also released appreciably higher levels of PGE2 compared with those of the nonstimulated control (Fig. 1C)Citation . That sPLA2IB-induced PGE2 release is caused by COX-2 activation is suggested by the marked inhibition of PGE2 production by the cells that were treated with NS398, a specific COX-2 inhibitor (data not shown).



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  		Fig. 1. Stimulation of COX-2 mRNA expression and PGE2 production by sPLA2IB. A, expression of COX-2 mRNA by Northern blotting. Total RNA (15 µg), prepared from cells treated without or with 50 nM sPLA2IB, were resolved by electrophoresis for Northern blot analysis using P32-labeled murine COX-2 cDNA probe. GAPDH probe was used to determine the quality and the amount of RNA loaded in each lane. Lane 1, untreated control; Lane 2, sPLA2-treated cells. B, Western blotting of cell lysates prepared from MC3T3E1 cells treated without (Lane 1) or with (Lane 2) 50 nM sPLA2IB for COX-2 as described in "Materials and Methods." C, the cells were incubated with 50 nM porcine pancreatic sPLA2IB for 3 and 6 h, respectively. The PGE2 concentrations in the culture medium were determined by ELISA. The results are the means of two independent determinations.

 
Analysis of the Mouse COX-2 Promoter Responsible for the sPLA2IB Stimulation.
To identify the regulatory element(s) responsible for sPLA2IB-stimulated activation of the COX-2 gene, we generated a series of promoter-luciferase fusion constructs containing varying lengths of 5' flanking sequences of the COX-2 gene (Fig. 2)Citation . Luciferase activities were measured in MC3T3E1 cells that were transiently transfected with each of these constructs and were treated with 50 nM porcine pancreatic sPLA2IB, and the results were compared with those of the untreated controls. As shown in Fig. 2Citation , although sPLA2IB treatment markedly increased the luciferase activity in cells that were transfected with the plasmid containing the full length COX-2 promoter (-963 to 70 bp), it is still ~25% of that observed with the truncation bearing the C/EBPß site. The fact that deletion of nucleotides -963 to -188 had enhanced luciferase activity compared with the intact promoter indicates the presence of apparent negative regulatory elements in this region. However, deletion of nucleotides ranging from -188 to -133 resulted in virtually a total lack of sPLA2IB-induced luciferase activity, suggesting the presence of critical regulatory elements in this region. The 5' flanking region of the COX-2 gene has been reported to contain the following regulatory elements: muscle-specific DNA-binding protein, MEF-2 (-490 to -481); NF-{kappa}B (-401 to -393); SP1 (-239 to -234); AP2 (-150 to -142); C/EBPß (-138 to -130); cyclic AMP response element (-56 to -51); and the TATA box (-30 to -25). The cells transfected with the construct containing the TATA box (-30 to -25) showed a basal level of luciferase activity similar to those found in cells transfected with the control construct, pGL-Basic.



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  		Fig. 2. Luciferase assay and COX-2 promoter analysis. Left, a diagrammatic representation of the 5'-flanking region of the COX-2 gene (-963 to 70 bp) containing several putative regulatory elements. Each deletion construct was ligated to a promoterless luciferase gene in an expression vector, pGL-Basic. Numbers indicate distance in bp from the start of transcription. The plasmids were used for transfection of the MC3T3E1 cells as described in "Materials and Methods." After incubating with 50 nM sPLA2IB for 6 h, the luciferase activity was measured in the cell lysates. The results of luciferase assay were normalized with total protein concentration in the cell lysates. Data are means of three determinations; bars, SE.

 
Identification of a Specific Regulatory Element Critical for sPLA2R-mediated COX-2 Gene Expression.
To further establish the identity of the critical response element, we prepared three double-stranded oligonucleotide probes (Fig. 3A)Citation : AN-1 (-151 to -121 bp) that contained AP2 and C/EBPß sites; and AN-2 and AN-3, which were specific for mutant AP2 and C/EBPß, respectively. The results of the mobility shift assay using nuclear extracts identified several broad, shifted bands (Fig. 3B)Citation in sPLA2IB-stimulated cells that interacted with the oligonucleotide probes AN-1 and AN-2, respectively. When nuclear extracts of untreated cells were used, only very faint bands were visible (Fig. 3B)Citation . Interestingly, the nuclear extracts of sPLA2IB-treated and untreated cells could not form protein-DNA complexes with the probe AN-3, the nucleotide sequence of which is identical to that of AN-1, except that it contained the sequence of the mutant C/EBPß motif (Fig. 3B)Citation . This result suggests that C/EBPß, but not AP2, is critical for the up-regulation of COX-2 gene expression by the sPLA2IB-receptor mediated pathway. Similar results were obtained when the antibody-mediated supershift assays were performed. A supershift of the bands occurred specifically in the presence of an antibody directed against C/EBP-ß (Fig. 3C)Citation . A faint supershifted band was also observed when the C/EBP{delta} antibody was used. However, other antibodies such as anti-C/EBP{alpha}, -AP2, -NF-{kappa}B p50, and NF-{kappa}B p65 did not cause any appreciable supershift (Fig. 3C)Citation .



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  		Fig. 3. Gel shift assay to identify a regulatory element involved in sPLA2R-mediated stimulation of COX-2 expression. A, the nucleotide sequences of double-stranded oligonucleotide probes synthesized: AN-1, -155 to -121 bp, including AP2 and C/EBPß sites; AN-2, the same as AN-1 except that the AP2 site was mutated; AN-3, containing mutations at the C/EBPß site. B, the electrophoretic mobility shift assay by using nuclear extracts prepared from MC3T3E1 cells treated without (Lanes 2, 5, and 8) or with (Lanes 3, 6, and 9) 50 nM sPLA2IB. Probes were incubated with nuclear extracts (2.5 µg) for 40 min at 30°C before applying onto a 4% nondenaturing polyacrylamide gel for electrophoresis. Arrows, shifted bands. Lanes 1, 4, and 7 are controls without the nuclear extract. C, electrophoretic mobility supershift by various antibodies. Lane 1, no antibody; Lanes 2, 3, and 4, antibodies against C/EBP{alpha}, C/EBPß, and C/EBP{delta}, respectively; Lane 5, anti-NF-{kappa}B p50; Lane 6, anti-NF-{kappa}B p65; Lane 7, AP-2. Note the clear supershifted band (arrow) in Lane 3 only.

 
C/EBPß Regulates the Receptor-mediated, sPLA2IB-induced COX-2 Gene Expression.
To unequivocally establish that C/EBPß is the critical response element, we performed site-specific mutagenesis of the region -188 to 70, which included coding regions of AP-2 and C/EPBß, respectively (Fig. 4)Citation . The response elements AP2 (CCGCTGCGG) and C/EBPß (TTGCGCAAC) were mutated to (CCGCTttGG) and (TgGaaCAAC), respectively. The MC3T3E1 cells, transfected with either the wild-type or the mutant constructs, were treated with 50 nM porcine pancreatic sPLA2IB, and the luciferase activities were determined. The results (Fig. 4)Citation showed that although mutation in the AP2 site did not alter the ability of sPLA2IB to transactivate the luciferase gene expression, such stimulation was totally ineffective when the cells were transfected with the construct containing the mutated C/EBPß sequence. Taken together, these results, for the first time, establish that C/EBPß, but not AP2, is the response element that is critical for the sPLA2R-mediated transcriptional activation of the COX-2 gene. Thus, the results of our present investigation, for the first time, uncovered the essential role of a specific transcription factor, C/EBPß, in the sPLA2IB-induced, receptor-mediated transcriptional activation of the COX-2 gene.



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  		Fig. 4. Mutation analysis of AP2 and C/EBPß sites. The wild-type and mutant plasmids, as indicated by an X over the sequence, were used for the transient transfection of MC3T3E1 cells by the LipofectAMINE method, as described in "Materials and Methods." When confluent, the cells were stimulated with 50 nM sPLA2IB for 6 h. The luciferase activity in cell lysates was measured and normalized with total protein concentration in the cell lysate. Data are means of two separate measurements; bars, SE.

 
Expression of cPLA2, sPLA2IB mRNA, sPLA2IIA mRNA, and sPLA2V mRNA in NIH3T3 and MC3T3E1 Cells.
COX-1 and COX-2 are the key enzymes that catalyze the production of PGs from AA. However, the specific PLA2 that is responsible for supplying the COX-2 substrate, AA, for the production of PGs in NIH3T3 and MC3T3E1 cells is unknown. Thus, to determine which PLA2(s) is responsible for generating AA for PG production, we studied the expression of cPLA2 mRNA, sPLA2IB mRNA, sPLA2IIA mRNA, and sPLA2V mRNA by Northern blotting (Fig. 5)Citation , using total RNA extracted from these cells. Interestingly, although in both of these cell lines the expression of sPLA21B mRNA, sPLA2IIA mRNA, and sPLA2V mRNA are totally lacking, the cPLA2 mRNA is readily detectable (Fig. 5)Citation These results indicate that cPLA2 is the most likely enzyme responsible for the generation of AA that is used by COX-2 in synthesizing PGs. Taken together, our results identify a specific PLA2 in NIH3T3 and MC3T3E1 cells that is vital for PG production in these cells.



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  		Fig. 5. Expression of different PLA2 mRNAs in NIH3T3 and MC3T3E1 cells. Total RNA (30 µg) was resolved by electrophoresis and blotted, as described in "Materials and Methods." They were hybridized with 32P-labeled murine sPLA2IB-, sPLA2IIA-, sPLA2V-, and cPLA2-specific cDNA probes. ß-Actin was used to determine the quality and the amount of RNA loaded in each lane. Note that although the cPLA2 signal is clearly detectable, the sPLA2 signals are not.

 
Coexpression of COX-2 and cPLA2 in Vivo.
To determine whether cPLA2 mRNA is coexpressed with COX-2 in vivo, we performed Northern blotting experiments using total RNA from several mouse tissues. Interestingly, the tissues that express COX-2 mRNA also express cPLA2 mRNA (Fig. 6)Citation , whereas the tissues in which COX-2 mRNA expression is undetectable (e.g.,. the pancreas), cPLA2 mRNA expression is also lacking. These results may indicate that COX-2 gene expression is closely associated with cPLA2 expression, and that cPLA2 may be instrumental in generating the substrate of COX-2 (i.e., AA) for PG production in vivo.



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  		Fig. 6. Coexpression of murine COX-2 and cPLA2 mRNAs in different organs. Two µg of poly(A)+ RNA were subjected to Northern blot analysis. The same blot was used for hybridization at 68°C for 2 h with 32P-labeled murine COX-2, cPLA2, and ß-actin cDNA probes. ß-Actin was used to determine the quality and the amount of RNA loaded in each lane. Note that both cPLA2 and COX-2 mRNAs are not detectable in both liver and in the pancreas, whereas in the heart, brain, lung, and spleen, both of these RNAs are readily detectable.

 
Effects of NSAIDs on COX-2 and cPLA2 mRNA Expression in NIH3T3 Cells.
As stated earlier, the mechanism by which NSAIDs reduce cancer mortality is not yet clear. It has been suggested that the cancer-chemopreventative effects of these drugs may not reside solely on their ability to inhibit COX-2 activity, because the dose that inhibits tumorigenesis is far greater than that which inhibits COX-2 catalysis. Because our results suggested that in NIH3T3 and MC3T3E1 cells cPLA2 may be the sole supplier of AA, we sought to determine the effects of NSAIDs on COX-2 as well as cPLA2 mRNA expression in these cells. It has been reported that the pancreatic sPLA2IB, via its receptor, can induce COX-2 and PG synthesis in mouse osteoblast, MC3T3E1 cells (20) . It has also been reported that 0.5–1.0 mM indomethacin stimulates the COX-2 mRNA and COX-2 protein production in mouse hepatic ML-457 cells (24) . Thus, we treated the NIH3T3 cells with 150 µM aspirin or sulindac for 6 h in the presence and absence of 50 nM porcine sPLA2I, and total RNA was isolated from the treated and untreated (control) cells. The results of Northern analysis reveal that sPLA2IB induces the synthesis of COX-2 mRNA (Fig. 7)Citation . The stimulation of the COX-2 mRNA by sPLA2IB and NSAIDs appears to be additive, and the stimulatory effects of sulindac were clearly more pronounced than those of aspirin.



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  		Fig. 7. Effects of NSAIDs on COX-2 mRNA expression in NIH3T3 cells. Cells (75–80% confluent) were treated with 50 nM sPLA2IB and 150 µM aspirin or sulindac (dissolved in DMSO), respectively, for 6 h in Opti-MEM 1 medium containing 5% FBS. Thirty µg of the total RNA were analyzed by Northern analysis as described In "Materials and Methods." 32P-Labeled murine COX-2 probe was used for hybridization at 65°C. GAPDH mRNA expression was determined to assess the quality and the amount of total RNA loaded in each lane.

 
It has been proposed that the pronounced suppression of PG production and cancer chemoprevention caused by NSAIDs may not be fully explained solely on the basis of their inhibitory effects on COX enzymatic activity. Thus, we investigated the effect of NSAIDs on the expression of cPLA2 because we have uncovered that NIH3T3 and MC3T3E1 cells do not express sPLA2IB mRNA, sPLA2IIA mRNA, or sPLA2V mRNA, and therefore, cPLA2 is most likely to be the supplier of AA. We found that treatment of the cells with either aspirin or sulindac caused drastic suppression of cPLA2 mRNA, and the effect of sulindac was much more pronounced than that of aspirin (Fig. 8)Citation . Taken together, our results suggest that NSAIDs inhibit PG production not only by inhibiting the catalytic activity of COXs, reported previously (1) , but also by severely restricting the supply of AA attributable to the suppression of cPLA2 mRNA expression. To our knowledge, this novel effect of NSAIDs on cPLA2 mRNA expression has not been reported previously.



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  		Fig. 8. Effect of aspirin and sulindac on the cPLA2 mRNA expression in NIH3T3 cells cultured in the absence (A) and presence (B) of sPLA2IB. NIH3T3 cells (75–80% confluent) were treated with 150 µM aspirin/sulindac in Opti-MEM 1 medium containing 5% FBS for 6 h. In a set of cultures, the cells were treated with 50 nM pancreatic sPLA2IB. After the incubation period, total cellular RNA was isolated. Thirty µg of the total RNA were resolved by agarose gel electrophoresis, blotted to the membrane, and hybridized at 68°C for 2 h with the 32P-labeled murine cPLA2 and ß-actin cDNA probes.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this investigation, we have uncovered that C/EBPß plays a critical role in sPLA2R-mediated transcriptional activation of the COX-2 gene expression in MC3T3E1 and NIH3T3 cells. In agreement with a previous report (24) , we found that NSAIDs stimulate sPLA2IB-mediated COX-2 mRNA expression. This is an unexpected finding, because it is well known that NSAIDs inhibit PG production (1) . Thus, the result of our present investigation, that NSAIDs drastically suppress cPLA2 mRNA while at the same time stimulating COX-2 mRNA expression, is novel. This latter finding suggests that NSAIDs possess more than one effect because it: (a) stimulates COX-2 mRNA and COX-2 protein production; (b) inhibits COX-2 catalytic activity, reported previously (1) ; and (c) suppresses cPLA2 mRNA expression.

It has been reported that sPLA2IB, via its receptor, regulates several important biological functions including cell proliferation (25, 26, 27) airway and vascular smooth muscle contraction (28 , 29) , chemokinesis (30) , and cellular invasion of the extracellular matrix (31) . Although the sPLA2R-mediated biological functions are increasingly becoming the focus of many investigations, the signaling pathway that leads to these cellular events remains poorly understood. Recently, sPLA2IB has been reported to transcriptionally activate the expression of the COX-2 gene in MC3T3E1 cells (20) . However, until now a specific transcription factor that mediates sPLA2IB-induced COX-2 gene expression has not been identified. One of the important findings of our present investigation is that C/EBPß plays a critical role in mediating the sPLA2IB-induced COX-2 gene activation in MC3T3E1 cells.

C/EBP comprises a diverse group of transcriptional regulators that influence tissue development and regeneration, inflammation, and intermediary metabolism (32 , 33) . These proteins belong to the basic leucine zipper family of transcription factors (33 , 34) . The first C/EBP proteins to be characterized were C/EBP{alpha} and C/EBPß (33 , 35 , 36) . They are primarily expressed in the adipose tissues, liver, and the intestinal tract (37) . Moreover, during an acute phase response, various isoforms of C/EBP, including C/EBPß and C/EBP{delta}, have been reported to be dramatically increased after bacterial lipopolysaccharide treatment of cells (38) .

COX-2 overexpression has been suggested to play an important role in increased metastatic potential in human colorectal cancer cells (reviewed in Ref. 6 ), and NSAIDs are shown to be chemopreventative for this cancer (7, 8, 9, 10, 11, 12, 13, 14) . In our present investigation, we sought to determine the specific transcription factor(s) responsible for sPLA2R-mediated COX-2 gene expression and to delineate the possible mechanisms underlying the effects of NSAIDs on this system. Our results uncovered two novel findings: (a) the critical role of C/EBPß in inducing sPLA2R-mediated transcriptional activation of the COX-2 gene; and (b) although the NSAIDs enhance the sPLA2IB-mediated COX-2 mRNA expression in MC3T3E1 and NIH3T3 cells, they drastically suppress the cPLA2 mRNA production. Moreover, these cells lack sPLA2IB mRNA, sPLA2IIa mRNA, and sPLA2V mRNA expression. These results suggest that although NSAID treatment increases COX-2 mRNA production, it may still suppress PG synthesis by depriving COX-2 of its substrate, AA, by drastically suppressing cPLA2 mRNA expression. Thus, NSAIDs inhibit PG production by their dual action on COX-2 as well as on cPLA2. It should be noted, however, that in variance with our results, Xu et al. (39) have reported recently that in vascular endothelial cells and in foreskin fibroblasts, aspirin and sodium salicylate suppress COX-2 gene transcription. This discrepancy between the results of our present study and those of Xu et al. (39) may be explained on the basis of the different cell types used in the two investigations.

There is considerable evidence to suggest that COX-2 plays important role(s) in tumorigenesis. It has been reported that COX-2 is up-regulated in transformed cells (40, 41, 42) and in various forms of cancer (43, 44, 45) . Inhibition of COX-2 activity caused a marked reduction in intestinal tumorigenesis (10) in a murine model of familial adenomatous polyposis that carried adenomatous polyposis coli (APC) gene mutation, found in a majority of colorectal cancers (46, 47, 48) . Similarly, inhibition of COX-2 gene expression caused virtually a complete suppression of colon cancer induced by azoxymethane (49) . It is widely accepted that cancer is attributable to the accumulation of mutations in specific genes that regulate cell division, DNA repair, and programmed cell death (46, 47, 48) . Although the exact link between COX-2 overexpression and tumorigenesis is not yet clearly understood, several possible mechanisms have been proposed. Increased PG production has been reported to occur in several tumor types (50, 51, 52, 53, 54) . In addition, PGs promote angiogenesis (55) , stimulate growth of malignant cells by accelerated cell proliferation (56) , and inhibit immune surveillance (57) . Furthermore, COX-2 gene overexpression has been reported to inhibit programmed cell death and increase the metastatic potential of human colorectal cancer cells (9 , 58) . Interestingly, these effects are reversed by NSAIDs such as sulindac. Thus, it has been suggested that suppression of COX-2 gene expression or inhibition of its catalytic activity may be an effective means to prevent cancer mortality (7, 8, 9, 10, 11, 12, 13, 14 , 59 , 60) . The fact that the dose of NSAIDs required to inhibit tumorigenesis is far greater than that required for inhibition of COX-2 catalysis suggests the existence of additional mechanisms of action of these drugs. Our present investigation provides an insight into the transcriptional regulation of COX-2 gene expression and demonstrates a novel molecular mechanism by which NSAIDs make COX-2 ineffective for PG production by depriving this enzyme of its substrate, AA. This substrate deprivation appears to be achieved by the striking suppression of cPLA2 mRNA expression by NSAIDs. To our knowledge, this is the first report demonstrating that NSAIDs suppress cPLA2 mRNA expression.

In an in vivo model of inflammation, it has been demonstrated recently that increased PG production is a two-component response: (a) increased COX-2 expression; and (b) increased supply of AA (61) . Moreover, cPLA2 preferentially catalyzes the release of AA from membrane phospholipids, and this enzyme is translocated from the cytosol to the nuclear membrane in a calcium-dependent manner (62, 63, 64) . Several studies have suggested that the cPLA2 is involved not only in the immediate phase but also in the delayed phase of eicosanoid generation (65) . The membrane association of COX-2 and its colocalization with cPLA2 have been reported. Moreover, it is established that COX-2 is the dominant isoform that mediates the delayed phase of PG production (66, 67, 68, 69) . Taken together, these results support the notion that a cPLA2/COX-2 coupled pathway is responsible for the delayed PG generation. It is possible that sustained PG generation is critical for the promotion phase in the multistep genetic process of tumorigenesis. Our results suggest that NSAIDs may not only inhibit the immediate phase but also the delayed phase of PG production reported in cancer cells (66, 67, 68, 69) . Because both AA and PGs regulate many vital functions, such as motility and invasion of the extracellular matrix, characteristic of most cancer cells, and have profound effects on cellular differentiation, we propose that the reduction of cancer mortality and the cancer chemopreventative effects of NSAIDs may, at least in part, stem from their ability to inhibit both COX-2 enzymatic activity and AA production by the suppression of cPLA2 mRNA expression.


   ACKNOWLEDGMENTS
 
We thank Drs. I. Owens, J. Y. Chou, J. DeBrun Butler, and S. W. Levin for critical review of the manuscript and valuable suggestions. We also thank Nadia Wang for performing DNA sequencing on mutant plasmids.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 C-J. Y. and A. K. M. contributed equally to this work. Back

2 To whom requests for reprints should be addressed, at NIH, Building 10, Room 9S241, Bethesda, MD 20892-1830. Phone: (301) 496-7213; Fax: (301) 402-6632; E-mail: mukherja{at}exchange.nih.gov Back

3 The abbreviations used are: COX, cyclooxygenase; PG, prostaglandin; NSAID, nonsteroidal anti-inflammatory drug; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; sPLA2, soluble PLA2; sPLA2R, sPLA2 receptor; C/EBP, CCAAT/enhancer-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AA, arachidonic acid; NF-{kappa}B, nuclear factor-{kappa}B; PMSF, phenylmethylsulfonyl fluoride. Back

Received 6/30/99. Accepted 12/14/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Vane J. R. Towards better aspirin. Nature (Lond.), 367: 215-216, 1994.[Medline]
  2. Meade E. A., Smith W. L., DeWitt D. L. Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-inflammatory drugs. J. Biol. Chem., 268: 6610-6614, 1993.[Abstract/Free Full Text]
  3. Smith W. L., Garavito R. M., DeWitt D. L. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J. Biol. Chem., 271: 33157-33160, 1996.[Free Full Text]
  4. Gierse J. K., Hauser S. D., Creely D. P., Koboldt C., Rangwala S. H., Isakson P. C., Seibert K. Expression and selective inhibition of the constitutive and inducible forms of cyclo-oxygenase. Biochem. J., 305: 479-484, 1995.
  5. DeWitt D. L., Smith W. L. Yes, but do they still get headaches?. Cell, 83: 345-348, 1995.[Medline]
  6. Prescott S. M., White R. L. Self-promotion? Intimate connections between APC and prostaglandin H synthase. Cell, 87: 783-786, 1996.[Medline]
  7. Thun M. J., Namboodiri M. M., Heath C. J. N. Aspirin use and reduced risk of fatal colon cancer. N. Engl. J. Med., 325: 1593-1596, 1991.[Abstract]
  8. Giovannucci E., Egan K. M., Hunter D. J., Stampfer M. J., Colditz G. A., Willett W. C., Speizer F. E. Aspirin and the risk of colorectal cancer in women. N. Engl. J. Med., 333: 609-614, 1995.[Abstract/Free Full Text]
  9. Boolbol S. K., Danenberg A. J., Chadburn A., Martucci C., Guo X., Ramonetti J. T., Areu-Goris M., Newmark H. L., Lepkin M. L., DeCosse J. J., Bertagnolli M. M. Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of FAP. Cancer Res., 56: 2556-2560, 1996.[Abstract/Free Full Text]
  10. Oshima M., Dinchuk J. E., Kargman S. L., Oshima H., Hancock B., Kwong E., Trzakos J. M., Evans J. F., Taketo M. M. Suppression of intestinal polyposis in APC{Delta}716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell, 87: 803-809, 1996.[Medline]
  11. Sheng G. G., Shao J., Sheng H., Hooton E. B., Isakson P. C., Morrow J. D., Coffey R. J., DuBois R. N., Beauchamp R. D. A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells. Gastroenterology, 113: 1883-1891, 1997.[Medline]
  12. Elder D. J. E., Paraseva C. NSAIDs to prevent colorectal cancer: a question of sensitivity. Gastroenterology, 113: 1999-2008, 1997.[Medline]
  13. Elder D. J. E., Paraskeva C. COX-2 inhibitors for colorectal cancer. Nat. Med., 4: 392-393, 1998.[Medline]
  14. Giardiello F. M., Hamilton S. R., Krush A. J., Piantadosi S., Hylind L. M., Celano P., Booker S. V., Robinson C. R., Offerhaus G. J. Treatment of colonic and rectal adenomas with sulindac in familial adenomatous polyposis. N. Engl. J. Med., 328: 1313-1316, 1993.[Abstract/Free Full Text]
  15. Waite, M. In: D. J. Hanahan (ed.), The Phospholipases, pp. 1–332. New York: Plenum Publishing Corp., 1998.
  16. Murakami M., Nakatani Y., Atsumi G., Inoue K., Kudo I. Regulatory functions of phospholipase A2. Crit. Rev. Immunol., 17: 225-283, 1997.[Medline]
  17. Tischfield J. A. A reassessment of the low molecular weight phospholipase A2 gene family in mammals. J. Biol. Chem., 272: 17247-17250, 1997.[Free Full Text]
  18. Ishizaki J., Hanasaki K., Higashino K-I., Kishino J., Kikuchi N., Ohara O., Arita H. Molecular cloning of pancreatic group I phospholipase A2 receptor. J. Biol. Chem., 269: 5897-5904, 1994.[Abstract/Free Full Text]
  19. Lambeau G., Ancian P., Barhanin J., Lazdunski M. Cloning and expression of a membrane receptor for secretory phospholipase A2. J. Biol. Chem., 269: 1575-1578, 1994.[Abstract/Free Full Text]
  20. Tohkin M., Kishino J., Ishizaki J., Arita H. Pancreatic-type phospholipase A2 stimulates prostaglandin synthesis in mouse osteoblastic cells (MC3T3E1) via a specific binding site. J. Biol. Chem., 268: 2865-2871, 1993.[Abstract/Free Full Text]
  21. Kishino J., Ohara O., Nomura K., Kramer R. M., Hitoshi A. Pancreatic-type phospholipase A2 induces group II phospholipase A2 expression and prostaglandin biosynthesis in rat mesangial cells. J. Biol. Chem., 269: 5092-5098, 1994.[Abstract/Free Full Text]
  22. Cupillard L., Mulherkar R., Gomez N., Kadam S., Valentin E., Lazdunski M., Lambeau G. Both group IB and IIA secreted phospholipase A2 are natural ligands of the mouse 180-kDa M-type receptor. J. Biol. Chem., 274: 7043-7051, 1999.[Abstract/Free Full Text]
  23. Hanasaki K., Arita H. Characterization of a high affinity binding site for pancreatic-type phospholipase A2 in the rat. J. Biol. Chem., 267: 6414-6420, 1992.[Abstract/Free Full Text]
  24. Ledwith B. J., Pauley C. J., Wagner L. K., Rokos C. L., Alberts D. W., Manam S. Induction of cyclooxygenase-2 expression by peroxisome proliferators and non-tetradecanoylphorbol-12,13-myristate-type tumor promoters in immortalized mouse liver cells. J. Biol. Chem., 272: 3707-3714, 1997.[Abstract/Free Full Text]
  25. Kishino J., Tohkin M., Arita H. Proliferative effect of phospholipase A2 in rat chondrocyte via its specific binding sites. Biochem. Biophys. Res. Commun., 186: 1025-1031, 1992.[Medline]
  26. Kinoshita E., Hanada K., Itoh M., Kumagai T., Kajiyama G., Sujiama M. Growth of human pancreatic cancer cells, induced by human pancreatic phospholipase A2, is mediated via its specific receptor but not via its catalytic property. Int. J. Oncol., 9: 1219-1225, 1996.
  27. Hanada K., Kinoshita E., Hirata M., Kajiyama G., Sujiyama M. Human pancreatic phospholipase A2 stimulates the growth of human pancreatic cancer cell line. FEBS Lett., 373: 85-87, 1995.[Medline]
  28. Sommers C. D., Bobbitt J. L., Bemis K. G., Snydu D. W. Porcine pancreatic phospholipase A2-induced contraction of guinea pig lung pleural strips. Eur. J. Pharmacol., 216: 87-96, 1992.[Medline]
  29. Nakajima M., Hanasaki K., Ueda M., Arita H. Effect of pancreatic-type phospholipase A2 on isolated porcine cerebral arteries via its specific binding sites. FEBS Lett., 309: 261-264, 1992.[Medline]
  30. Kanemasa T., Hanasaki K., Arita H. Migration of vascular smooth muscle cells by phospholipase A2 via specific binding sites. Biochim. Biophys. Acta, 1125: 210-214, 1992.[Medline]
  31. Kundu G. C., Mukherjee A. B. Evidence that porcine pancreatic phospholipase A2 via its high affinity receptor stimulates extracellular matrix invasion by normal and cancer cells. J. Biol. Chem., 272: 2346-2353, 1997.[Abstract/Free Full Text]
  32. Wedel A., Ziegler-Heibrock H. W. L. The C/EBP family of transcription factors. Immunobiology, 193: 171-185, 1995.[Medline]
  33. Poli V., Mancini F. P., Cortese R. IL-6 DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell, 63: 643-653, 1990.[Medline]
  34. Landschulz W. H., Johnson P. F., Adashi E. Y., Graves B. J., McKnight S. L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3–L1 cells. Genes Dev., 2: 785-795, 1988.
  35. Descombes P., Chojkier M., Lichtsteiner S., Falvey E., Schibler U. LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev., 4: 1541-1551, 1990.[Abstract/Free Full Text]
  36. Cao Z., Umek R. M., McKnight S. L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev., 5: 1538-1553, 1991.[Abstract/Free Full Text]
  37. Birkenmeier E. H., Gwynn B., Howard S., Jerry J., Gordon J. I., Landschultz W. H., McKnight S. L. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein. Genes Dev., 3: 1146-1156, 1989.[Abstract/Free Full Text]
  38. Alam T., Au M. R., Papaconstinon J. Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response. J. Biol. Chem., 267: 5021-5024, 1992.[Abstract/Free Full Text]
  39. Xu X. M., Sansores-Garcia L., Chen X. M., Matijevic-Aleksic N., Du M., Wu K. K. Suppression of inducible cyclooxygenase-2 gene transcription by aspirin and sodium salicylate. Proc. Natl. Acad. Sci. USA, 96: 5292-5297, 1999.[Abstract/Free Full Text]
  40. Fujita T., Marsui M., Takaku K., Uetake H., Ichikawa W., Taketo M., Sugihara K. Size- and invasion-dependent increase in cyclooxygenase-2 levels in human colorectal carcinomas. Cancer Res., 58: 4823-4826, 1998.[Abstract/Free Full Text]
  41. Subbaramaiah K., Telang N., Ramonetti J. T., Araki R., DeVito B., Weksler B. B., Dannenberg A. J. Transcription of cyclooxygenase-2 is enhanced in transformed mammary epithelial cells. Cancer Res., 56: 4424-4429, 1996.[Abstract/Free Full Text]
  42. Subbaramiah K., Chung W. J., Michaluart P., Telang N., Tanabe T., Inoue H., Jang M., Pezzuto J. M., Dannenberg A. J. Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J. Biol. Chem., 273: 21875-21882, 1998.[Abstract/Free Full Text]
  43. Ristimaki A., Honkanen N., Jankala H., Sipponen P., Harkonen M. Expression of cyclooxygenase-2 in human gastric carcinoma. Cancer Res., 57: 1276-1280, 1997.[Abstract/Free Full Text]
  44. Parett M. L., Harris R. E., Joarder F. S., Ross M. S., Claussen K. P., Robertson F. M. Cyclooxygenase-2 gene expression in human breast cancer. Int. J. Oncol., 10: 503-507, 1997.
  45. Kargman S., O’Neill G., Vickers P., Evans J., Mancini J., Jothy S. Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer. Cancer Res., 55: 2556-2559, 1995.[Abstract/Free Full Text]
  46. Vogelstein B., Fearson E. R., Hamilton S. R., Kern S. E., Preisinger A. C., Leppert M., Nakamura Y., White R., Smiths A. M. M., Bos J. L. Genetic alterations during colorectal-tumor development. N. Engl. J. Med., 319: 525-532, 1988.[Abstract]
  47. Fearson E. R. Human cancer syndromes: clues to the origin and nature of cancer. Science (Washington DC), 278: 1043-1050, 1997.[Abstract/Free Full Text]
  48. Kinzler K. W., Vogelstein B. Lessons from hereditary colorectal cancer. Cell, 87: 159-170, 1996.[Medline]
  49. Kawamori T., Rao C. V., Seibert K., Reddy B. S. Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res., 58: 409-412, 1998.[Abstract/Free Full Text]
  50. Bennett A. The production of prostanoids in human cancers and their implications for tumor progression. Prog. Lipid Res., 25: 539-542, 1986.[Medline]
  51. Rolland P. H., Martin P. M., Jacquemier J., Rolland A. M., Toga M. Prostaglandin in human breast cancer: evidence suggesting that an elevated prostaglandin production is a marker of high metastatic potential for neoplastic cells. J. Natl. Cancer Inst., 64: 1061-1070, 1980.
  52. Jung T. T. K., Berlinger N. T., Juhn S. K. Prostaglandins in squamous cell carcinoma of the head and neck: a preliminary study. Laryngoscope, 95: 307-312, 1985.[Medline]
  53. Lupulescu A. Prostaglandins, their inhibitors and cancer. Prostaglandins Leukot. Essent. Fatty Acids, 54: 83-94, 1996.[Medline]
  54. Zimmermann K. C., Sabia M., Weber A., Borchard F., Gabbert H. E., Sehror K. Cyclooxygenase-2 expression in human esophageal carcinoma. Cancer Res., 59: 198-204, 1999.[Abstract/Free Full Text]
  55. Ben-Av P., Crofford L. J., Wilder R. L., Hla T. Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS Lett., 372: 83-87, 1995.[Medline]
  56. Sheng H., Shao J., Morrow J. D., Beauchamp R. D., DuBois R. N. Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res., 58: 362-366, 1998.[Abstract/Free Full Text]
  57. Goodwin J. S., Ceuppens J. Regulation of immune response by prostaglandins. J. Clin. Immunol., 3: 295-314, 1983.[Medline]
  58. Tsujii M., DuBois R. N. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell, 83: 493-501, 1995.[Medline]
  59. Williams C. S., Smalley W., DuBois R. N. Aspirin use and potential mechanisms for colorectal cancer prevention. J. Clin. Investig., 100: 1325-1329, 1997.[Medline]
  60. Mestre J. R., Subbaramaiah K., Sacks P. G., Schantz S. P., Tanabe T., Inoue H., Dannenberg A. J. Retinoids suppress phorbol ester-mediated induction of cyclooxygenase-2. Cancer Res., 57: 1081-1085, 1997.[Abstract/Free Full Text]
  61. Hamilton L. C., Mitchell J. A., Tomlinson A. M., Warner T. D. Synergy between cyclooxygenase-2 induction and arachidonic acid supply in vivo: consequences for nonsteroidal antiinflammatory drug efficacy. FASEB J., 13: 245-251, 1999.[Abstract/Free Full Text]
  62. Peters-Golden M., McNish R. W. Redistribution of lipoxygenase and cytosolic phospholipase A2 to the nuclear fraction upon macrophage activation. Biochem. Biophys. Res. Commun., 196: 147-153, 1993.[Medline]
  63. Glover S., de Carvalho M. D., Bayburt T., Jonas M., Chi E., Leslie C. C., Gelb M. H. Translocation of the 85 kDa phospholipase A2 from cytosol to the nuclear envelope in rat basophilic leukemic cells stimulated with calcium ionophore or IgE/antigen. J. Biol. Chem., 270: 15359-15365, 1995.[Abstract/Free Full Text]
  64. Schievella A. R., Regier M. K., Smith W. L., Lin L. L. Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum. J. Biol. Chem., 270: 30749-30756, 1995.[Abstract/Free Full Text]
  65. Morita I., Schindler M., Regier M. K., Otto J. C., Hori T., DeWitt D. L., Smith W. L. Different intracellular locations for prostaglandin endoperoxidase H synthase-1 and -2. J. Biol. Chem., 270: 10902-10908, 1995.[Abstract/Free Full Text]
  66. Murakami M., Matsumoto R., Austen K. F., Arm J. P. Prostaglandin endoperoxide synthase-1 and -2 couple to different transmembrane stimuli to generate prostaglandin D2 in mouse bone marrow-derived mast cells. J. Biol. Chem., 269: 22269-22276, 1994.[Abstract/Free Full Text]
  67. Meade E. A., Smith W. L., DeWitt D. L. Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal antiinflammatory drugs. J. Biol. Chem., 268: 6610-6615, 1993.
  68. Mitchell J. A., Akarasereenont P., Thiemermann C., Flower R. J., Vane J. R. Selectivity of nonsteroidal anti-inflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase. Proc. Natl. Acad. Sci. USA, 90: 11693-11699, 1994.[Abstract/Free Full Text]
  69. Peri K. G., Hardy P., Li D. Y., Varma D. R., Chemtob S. Prostaglandin G/H synthase-2 is a major contributor of brain prostaglandin in the newborn. J. Biol. Chem., 270: 24615-24651, 1995.[Abstract/Free Full Text]



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S. Beck, G. Lambeau, K. Scholz-Pedretti, M. H. Gelb, M. J. W. Janssen, S. H. Edwards, D. C. Wilton, J. Pfeilschifter, and M. Kaszkin
Potentiation of Tumor Necrosis Factor {alpha}-induced Secreted Phospholipase A2 (sPLA2)-IIA Expression in Mesangial Cells by an Autocrine Loop Involving sPLA2 and Peroxisome Proliferator-activated Receptor {alpha} Activation
J. Biol. Chem., August 8, 2003; 278(32): 29799 - 29812.
[Abstract] [Full Text] [PDF]

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 J. Biol. Chem.Home page
F. G. Bottone Jr., J. M. Martinez, J. B. Collins, C. A. Afshari, and T. E. Eling
Gene Modulation by the Cyclooxygenase Inhibitor, Sulindac Sulfide, in Human Colorectal Carcinoma Cells: POSSIBLE LINK TO APOPTOSIS
J. Biol. Chem., July 3, 2003; 278(28): 25790 - 25801.
[Abstract] [Full Text] [PDF]

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 Clin. Cancer Res.Home page
H. A. Minter, J. W. Eveson, S. Huntley, D. J. E. Elder, and A. Hague
The Cyclooxygenase 2-selective Inhibitor NS398 Inhibits Proliferation of Oral Carcinoma Cell Lines by Mechanisms Dependent and Independent of Reduced Prostaglandin E2 Synthesis
Clin. Cancer Res., May 1, 2003; 9(5): 1885 - 1897.
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Home page
 J. Immunol.Home page
D.-W. Park, J.-R. Kim, S.-Y. Kim, J.-K. Sonn, O.-S. Bang, S.-S. Kang, J.-H. Kim, and S.-H. Baek
Akt as a Mediator of Secretory Phospholipase A2 Receptor-Involved Inducible Nitric Oxide Synthase Expression
J. Immunol., February 15, 2003; 170(4): 2093 - 2099.
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 Molecular Cancer TherapeuticsHome page
J. Chu, F. L. Lloyd, O. C. Trifan, B. Knapp, and M. T. Rizzo
Potential Involvement of the Cyclooxygenase-2 Pathway in the Regulation of Tumor-associated Angiogenesis and Growth in Pancreatic Cancer
Mol. Cancer Ther., January 1, 2003; 2(1): 1 - 7.
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V. Blais, J. Zhang, and S. Rivest
In Altering the Release of Glucocorticoids, Ketorolac Exacerbates the Effects of Systemic Immune Stimuli on Expression of Proinflammatory Genes in the Brain
Endocrinology, December 1, 2002; 143(12): 4820 - 4827.
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H. Q. Wang, M. P. Kim, H. F. Tiano, R. Langenbach, and R. C. Smart
Protein Kinase C-{alpha} Coordinately Regulates Cytosolic Phospholipase A2 Activity and the Expression of Cyclooxygenase-2 through Different Mechanisms in Mouse Keratinocytes
Mol. Pharmacol., April 1, 2001; 59(4): 860 - 866.
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E. Heiss, C. Herhaus, K. Klimo, H. Bartsch, and C. Gerhauser
Nuclear Factor kappa B Is a Molecular Target for Sulforaphane-mediated Anti-inflammatory Mechanisms
J. Biol. Chem., August 17, 2001; 276(34): 32008 - 32015.
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